基于双重配体/受体结合效应靶向淋巴结DCs的肿瘤疫苗构建及机制研究

The application of nanoparticle-based adjuvants to therapeutic cancer vaccine, which could active target and activate dendritic cells (DCs) and then induce T cell immune response, has become a research hotspot in the field of cancer immunotherapy. We found that the type and intensity of immune response induced by DCs targeting cancer vaccine is closely related to the location and number of the activated DCs. The current study mainly focused on the peripheral tissue DCs rather than the most effective lymph nodes (LNs) tissue, leading to poor vaccine efficacy and reproducibility. It has been predicted that active targeting and activation of DCs in LNs is advantageous to the induction of memory T cell immune response and enhancement of the antigen specific immune response, which can play a more effective role in antitumor therapy. However, this theory needs to be confirmed. In this project, Trp2/A/MART-1 peptide vaccine will be constructed, which was mediated by hyaluronic acid/lymphatic endothelial receptor and CCL21/CCR7 dual ligand/receptor binding effect, active targeting and activation of DCs in vice cortex of LNs and cross-presenting function. At the same time, the innovative technology of active targeted activation of DCs in LNs was established. This study will verify the rationality of the design and construction of nanopartical-based peptide vaccine and clarify the mechanism of peptide vaccine enhancing peptide immune effect by in vitro and in vivo evaluating. It will provide experimental basis and theoretical basis for the establishment of safe and efficient nanoparticle-based vaccine adjuvant and tumor peptide vaccine. The aim of the project is to confirm the scientific hypothesis and to lay a foundation for in-depth study of the tumor vaccine and action mechanism of active targeting and activation of DCs in LNs, which is of important scientific significance and research value.

纳米佐剂用于治疗性肿瘤疫苗，主动靶向激活树突状细胞（DCs），诱导T细胞免疫应答，已成为肿瘤免疫治疗领域的研究热点。申请者发现：DCs靶向肿瘤疫苗诱导的免疫反应类型和强度与激活DCs的位置和数量密切相关。目前研究主要针对靶向外周组织DCs，而不是引起免疫反应最有效的淋巴结（LNs）组织DCs，导致肿瘤疫苗效应性和重现性较差。理论预测主动靶向激活LNs内DCs，有利于诱导T细胞记忆性免疫应答，并增强抗原特异性免疫应答，更有效发挥抗肿瘤作用，但尚需进一步证实。本研究旨在构建透明质酸/淋巴管内皮受体和CCL21/CCR7双重配体/受体结合效应介导，主动靶向激活LNs副皮质区DCs，并具有促进抗原交叉提呈功能的Trp2/A/MART-1纳米多肽疫苗；建立主动靶向激活LNs内DCs的创新技术。通过对纳米多肽疫苗进行体内外评价，验证其设计构建合理性，并阐明其增强多肽免疫效应的作用机制，为建立安全、高效的纳米疫苗佐剂和多肽疫苗提供实验基础和理论依据。本研究旨在证实科学假说，为深入研究主动靶向激活LNs内DCs的肿瘤疫苗及作用机制奠定基础。纳米疫苗佐剂结合肿瘤多肽疫苗研究具有重要的科学意义和研究价值。

纳米佐剂应用于治疗性肿瘤疫苗，主动靶向激活树突状细胞（DCs），诱导T细胞免疫应答，已成为肿瘤免疫治疗领域的研究热点。DCs靶向的肿瘤疫苗诱导免疫反应的类型和强度与激活DCs位置和数量密切相关。目前的研究主要针对外周组织DCs，而不是引起免疫反应最有效的淋巴结（LNs）组织中DCs，导致疫苗的效应性和重现性较差。本研究拟构建靶向淋巴结组织，并具有内涵体逃逸作用的阳离子脂质体，以人乳头瘤病毒16 E7多肽（HPV 16 E7）联合CpG寡聚核苷酸（CpG ODN）佐剂为模型药物，以C57BL/6小鼠皮下移植肺上皮细胞（TC-1）为宫颈癌模型，考察该疫苗传递系统增强机体免疫应答，提高抗肿瘤活性的作用。本研究成功合成脂质材料2-羧基环己烷-1-羰基-透明质酸-二肉豆蔻酰基磷脂酰乙醇胺（CHex-HA-DMPE），以1H-NMR验证中间体CHex-HA的取代度；酸碱滴定法测定CHex66-HA的pKa值为6.70；以红外光谱中酰胺羰基和酯羰基的特征峰位移确证CHex-HA-DMPE成功合成。以CHex-HA-DMPE为主要载体材料制备阳离子脂质体，粒径约160 nm，Zeta电位+55.63 mV。以DID为探针对小鼠淋巴结组织成像及冰冻切片结果显示，透明质酸（HA）修饰的脂质体组，小鼠淋巴结荧光强度显著高于不含HA脂质体组及对照组，证明其具有淋巴结靶向作用。脂质体对HPV 16 E7和CpG ODN的包封率分别为45.87%和67.50%。抑瘤实验表明，载体递送HPV 16 E7和CPG ODN（CCHL E7/CpG）对小鼠肿瘤具有明显抑制作用，各组小鼠体重和生化指标与健康小鼠无显著差异，主要脏器HE染色结果均显示正常。qRT-PCR及免疫组化结果表明，与对照组和空白脂质体组相比，CCHL E7/CpG组IFN-γ、IL-10、iNOS表达显著上调，VEGF、C-myc、Bcl-2表达显著下调；并且CCHL E7/CpG组肿瘤组织及脾中CD8+ T细胞、CD4+ T细胞数量显著上调。本研究成功合成HA修饰，且具有pH敏感性的脂质材料，以其构建的多功能脂质体具有将多肽疫苗和佐剂靶向递送至淋巴结的作用。多功能脂质体共传递HPV16 E7与CpG ODN，能够显著提高HPV16 E7对宫颈癌的免疫治疗作用，并在一定程度上逆转免疫抑制性肿瘤微环境，具有重要的科学意义和研究价值。